Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol152.Issue2.2000:

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A small gene family of broad bean codes for late nodulins

containing conserved cysteine clusters

Martin Fru¨hling, Ulrike Albus, Natalija Hohnjec, Gerhard Geise, Alfred Pu¨hler,

Andreas M. Perlick *

Uni6ersita¨t Bielefeld,Lehrstuhl fu¨r Genetik,Postfach100131,D-33501Bielefeld,Germany

Received 5 August 1999; received in revised form 25 October 1999; accepted 25 October 1999

Abstract

Five transcripts encoding different members of a nodulin family with conserved cysteine clusters (Cys-X4-Asp-Cys and

Cys-X4-Cys) were identified in broad bean root nodules. They displayed homologies to the early nodulins PsENOD3 and

PsENOD14 and the late nodulin PsNOD6 from pea. In addition to the occurence of putative secretory signal peptides, the spatial distribution of the cysteine residues was comparable in both the broad bean and the pea nodulins. Based on tissue print hybridizations, we found that the corresponding broad bean genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 were expressed in the interzone II – III and the nitrogen fixing zone III of mature nodules whereas the gene VfNOD-CCP4 was first induced in the prefixing zone II. A strong expression of the VfNOD-CCP2 gene only could be detected the interzone II – III region. Sequence analysis of a genomic VfNOD-CCP1 clone isolated revealed the presence of one intron seperating a first exon encoding the signal peptide from a second exon encoding the cysteine cluster domain of this nodulin. Apart from the multiple presence of the common nodulin motifs AAAGAT and CTCTT on both DNA strands of the putative VfNOD-CCP1 promoter region a sequence element resembling the organ specific element of the soybean lbc3 gene promoter was identified. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Symbiotic nitrogen fixation; Tissue print hybridization; Metal binding;Vicia fabaL.

www.elsevier.com/locate/plantsci

1. Introduction

The interaction of soil bacteria belonging to the

genera Rhizobium, Bradyrhizobium and Azorhizo

-bium with leguminous plants leads to the

forma-tion of a novel plant organ — the root nodule [2,23,43]. In mature nodules, nitrogen-fixation is carried out by the microsymbiont.

The elicitation and development of an effective symbiosis involves a mutual exchange of specific

signals between both partners [37] and is accompa-nied by the expression of specific genes in both rhizobia and their host plants [23,45]. Initially, nodules develop from primordia by differentiation of specialized tissues. The nodules are infected by the symbiotic bacteria via infection threads [18]. After their release from these tubular structures, the bacteria are enclosed by a modified plant-derived membrane (peribacteriod membrane) and differentiate into bacteriods capable of reducing atmospheric nitrogen. The central tissues of fully developed indeterminate nodules formed on tem-perate legumes display a characteristic zonation. An apical meristem is followed by the prefixing

zone II, the interzone II/III rich in amyloplasts,

the nitrogen fixing zone III, and the senescence zone IV, according to the nomenclature of Vasse et al. [44].

The sequence data reported will appear in the EMBL database under the accession numbers AJ243461 and AJ243462 (VfNOD-CCP1), AJ243463 (VfNOD-CCP2), AJ243464 (VfNOD-CCP3), AJ243465 (VfNOD-CCP4), and AJ243466 (VfNOD-CCP5), respec-tively.

* Corresponding author. Tel.: +49-521-106-5631; fax: + 49-521-106-5626.

E-mail address: [email protected] (A.M. Perlick)

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M.Fru¨hling et al./Plant Science152 (2000) 67 – 77

In recent years, the bacterial genetics of nodule formation and nitrogen fixation have been exten-sively studied [9,10]. An increasing number of plant genes specifically expressed in nodules, termed nodulin genes [42], have been identified and subdivided into early and late nodulin genes according to the time point of their expression [23,45]. Early nodulin genes (ENODs) are associ-ated with organogenesis and bacterial invasion of the root nodule. The sequences of many early nodulins (ENOD2 and ENOD12, for example) suggest that they represent proline-rich proteins most probably involved in cell wall biosynthesis. In general, late nodulin gene (NOD) products are thought to be involved in nodule function and include the oxygen transporter leghemoglobin,

en-zymes of carbon and nitrogen metabolism,

proteins located in the peribacteroid interface as well as a number of proteins, the functions of which remain to be identified [6].

To investigate the organ-specific gene expression

in broad bean (Vicia faba L.) root nodules, we

have constructed a nodule-specific cDNA library of approximately 700 independent cDNAs [28]. In addition to different leghemoglobins [12], tran-scripts homologous to the early nodulin genes

PsENOD2 [41], PsENOD5 and PsENOD12 [35]

were isolated [28]. We also detected a family of at least five nodule-specifically expressed broad bean

genes encoding glycine-rich proteins [20,36],

VfNOD28/32 the homologue ofMsNOD25 [19,21] and VfNOD32 encoding a narbonin-like nodulin with homologies to chitinases [29].

In this paper, we report on the characterization of five broad bean transcripts encoding a family of late nodulins with conserved cysteine-clusters. We describe the spatial and temporal expression of the transcripts in broad bean nodules and finally we report on the isolation and analysis of a corre-sponding genomic sequence.

2. Methods

2.1. Biological material, cDNA and genomic libraries

Broad bean plants (Vicia faba L. cv. Kleine

Thu¨ringer) were grown in the greenhouse in sterile clay granules (Seramis) using surface-sterilized seeds (saturated Ca-hypochlorite, 30 min).

Nodu-lated plants were obtained by inoculation of 2

day-old seedlings with Rhizobium leguminosarum

bv. 6_iciae _{VF39 [30]. Flowers and seeds were} analysed from field grown plants inoculated 3 days after sowing. The nodule cDNA library was

con-structed in lgt11 from poly(A)+ _{RNA isolated}

from root nodules of V. faba L. cv. Kleine

Thu¨ringer [28]. A broad bean genomic library was

prepared in lEMBL3 [11] according to Sambrook

et al. [33].

2.2. Library screening, isolation of nucleic acids and recombinant DNA techniques

Recombinant lgt11 and lEMBL3 phages were

plated and screened for positive clones as de-scribed previously [20]. Three rounds of screening were performed to obtain single positive plaques. Isolation of phage DNA was carried out using standard protocols [33]. Plasmid DNA was

iso-lated from E. coli XL1-Blue using the ‘Plasmid

Mini Kit’ (Qiagen) according to the manufactur-er’s instructions. Probe DNA was extracted from agarose gels using the ‘QIAEX Gel Extraction Kit’ (Qiagen). For Northern blotting, RNA was iso-lated from nodules (32 days after sowing), unin-fected roots (32 days after sowing), leaves (32 days after sowing), seeds (90 days after sowing), epi-cotyls (8 days after sowing), stems (12 days after sowing) and flowers (60 days after sowing) of

broad beans using standard protocols [5].

Poly(A)+ _{RNA fractions were isolated by one}

cycle of oligo(dT)-cellulose chromatography.

The ‘5%-AmpliFINDER RACE KIT’ (Clontech)

was used to perform 5% _{RACE reactions. The}

cDNA synthesis was carried out according to the manufacturer’s instructions using gene-specific

primers. For subsequent PCR amplification of 5%

cDNA fragments gene-specific nested primers were used. To generate overlapping sequencing clones, exonuclease III digestions were carried out using the ‘Double Stranded Nested Deletion Kit’ (Phar-macia) according to the manufacturer’s instruc-tions. All other in vitro DNA manipulations were carried out using standard protocols [33].

2.3. DNA sequencing and analysis

Sequencing reactions have been carried out ac-cording to Zimmermann et al. [46] using the ‘Au-toRead Sequencing Kit’ (Pharmacia). Sequencing

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gels were run on the ‘A.L.F. DNA Sequencer’ (Pharmacia) using sequencing gel mixes of stan-dard composition. All sequences reported here were determined from both strands. DNA se-quence data were read using the ‘A.L.F. MAN-AGER V3.0’ software (Pharmacia) and analysed using the programme ‘ANALYSEQ’ [38] and ‘LFASTA’ (based on [27]). Multiple sequence alignments were carried out using the program

‘CLUSTAL’ [14] from the PC/Gene software

package (IntelliGenetics, release 6.8). Predictions of signal peptides and their cleavage sites were carried out using the ‘SignalP world wide web server’ (release V1.1) [26].

2.4. Northern and cDNA-cDNA hybridizations

Northern blotting and hybridizations were car-ried out as described previously [28]. About 50 ng of probe DNA isolated from agarose gels were

labelled with 50 mCi of a32_{P-dATP according to}

Feinberg and Vogelstein [8]. Stringent washes were

carried out at room temperature using 2×SSC,

0.1% (w/v) SDS (5 min) and at 68°C using 0.2×

SSC, 0.1% (w/v) SDS (twice for 30 min each).

For cDNA-cDNA hybridizations, 0.2 mg EcoRI

digested DNA from different cDNA clones was separated electrophoretically and blotted onto Hy-bond-N nylon membranes (Amersham) using

stan-dard protocols [33]. Radioactively labeled

first-strand cDNA probes were synthesized from 1

mg poly(A)+ _{RNA according to Fru¨hling et al.}

[12] and used immediately for hybridization. Fil-ters were hybridized in a solution containing 50

mM Na phosphate pH 7.0, 5×SSC, 0.1% (w/v)

lauroylsarcosin, 2% (w/v) blocking reagent

(Boehringer), 7% (w/v) SDS and 50% (v/v)

for-mamide for 48 h at 42°C. Stringent washes were

carried out as described for Northern

hybridizations.

2.5. Tissue print hybridizations

Longitudinal sections of mature broad bean nodules (harvested 32 days after sowing) were printed on Hybond-N nylon membranes (Amer-sham) as described [36]. Hybridizations against digoxigenin-labeled antisense riboprobes were car-ried out as reported by Ku¨ster et al. [20]. Stringent washes and detection of hybridizing transcripts were carried out according to Kessler [17]. As a

control, prints were hybridized against the corre-sponding sense probes. In none of the cases ex-pression above background was observed.

To relate hybridizing regions to nodule zones, sections used for tissue-printing were stained for

starch in Lugol’s solution containing 1% (w/v) KI

and 1% (w/v) I2in distilled water. Stained sections

were photographed at the same magnification as the tissue-print filters.

3. Results

3.1. Fi6_{e broad bean cDNAs encode small}

polypeptides characterized by conser6ed cysteine clusters

Preliminary sequence data indicated that incom-plete cDNAs from five different clone groups of a broad bean nodule-specific cDNA library [28] en-coded polypeptides with conserved cysteine clus-ters (cysteine cluster proteins: CCPs) [12]. The corresponding genes were designated CCP1, CCP2, CCP3, VfNOD-CCP4 and VfNOD-CCP5, respectively. To isolate and determine transcript sequences covering the entire CCP coding regions, we rescreened a nodule cDNA library with appropriate probes. In addi-tion the RACE-PCR technique was used to com-plete cDNAs which did not extend to the

5%_{-untranslated transcript regions. The sequences}

of the different cDNAs and RACE fragments isolated for each of the five individual transcripts showed identity in the regions of overlap. There-fore it was reasonable to combine these sequences. The open reading frames of the full-length CCP transcript sequences presented encoded for re-markable small polypeptides. Their calculated molecular masses ranged from 6.67 kDa for VfNOD-CCP5 to 7.57 kDa for VfNOD-CCP3 (Table 1). An alignment of the deduced polypep-tide sequences is shown in Fig. 1. The comparison on the amino acid level revealed only limited sequence identities between 22.1% (VfNOD-CCP3 vs. VfNOD-CCP5) and 43.9% (VfNOD-CCP2 vs. VfNOD-CCP4). On the other hand, a bipartite domain structure of the CCPs was found to be well conserved. Analysis of the hydrophobic N-terminal polypeptide regions revealed characteris-tics associated with secretory signal peptides. According to Nielsen et al. [26] each of the five

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Table 1

Comparison of five broad bean nodulins with conserved cyteine clustersa

Primary Amino acid sequence identity to (%) Transcript Processed

polypeptide polypeptide

VfNOD-kDa VfNOD- VfNOD- VfNOD- VfNOD- ENOD3 ENOD14 NOD6 aa

kDa aa

CCP2 CCP3 CCP5

CCP1 CCP4

¯ 34.8 31.9 28.6 28.4 27.8

40 4.45 23.6 28.2

7.44 65

VfNOD-CCP1

4.12

62 6.98 37 34.8 ¯ 35.7 43.9 24.6 36.2 35.3 34.3

VfNOD-CCP2

31.9 35.7 ¯ 43.7 22.1 36.8

40 27.4

7.57 4.63 35.2

VfNOD- 66 CCP3

28.6 43.9 43.7 ¯ 30.3 54.3 38.8

VfNOD- 64 7.42 40 4.67 33.8

CCP4

28.4 24.6 22.1 30.3 ¯ 29.6 32.4

33 3.69 24.2

VfNOD- 59 6.67 CCP5

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CCPs meet all the criteria of a functional eukary-otic signal sequence. The cleavage sites were

pre-dicted to be between Ser25 _and _Thr26 _for

VfNOD-CCP1, between Ala25 _{and Gln}26 _for

VfNOD-CCP2, between Gly26 _{and Glu}27 _for

VfNOD-CCP3, between Ala24 _{and Asn}25 _for

VfNOD-CCP4 and between Ala26 _{and Ser}27 _for

VfNOD-CCP5. The most striking feature of the C-terminal polypeptide domain were four con-served cysteine residues. They were arranged in

two clusters of the form Cys-X4-Asp-Cys and

Cys-X4-Cys, respectively. As is evident from Fig. 1, the

distance between the cysteine clusters vary be-tween 9 amino acids within VfNOD-CCP1 and

VfNOD-CCP3 and 14 amino acids within

VfNOD-CCP4. From these results we concluded that the five CCP transcripts encode members of a small broad bean polypeptide family.

Database searches revealed homologies of the different CCPs to the early nodulins PsENOD3

and PsENOD14 [35] and the late nodulin

PsNOD6 [16] from pea (Fig. 1). We could not detect any other significant homologies between the broad bean CCPs and protein sequences in the databases. The homologous pea nodulins dis-played the same characteristics as the CCPs. In addition to the occurence of putative signal pep-tides, the spatial distribution of the conserved

cysteine residues was comparable. In some cases the amino acid identities between the CCPs and the pea nodulins exceeded the identities between the members of the broad bean polypeptide fam-ily. For example VfNOD-CCP4 and PsENOD3 matched in 54.3% of all amino acid residues (see Table 1).

3.2. The CCP transcripts identified are exclusi6_ely

expressed in root nodules

Our previous analysis showed that the CCP transcripts were expressed strongly in broad bean root nodules [12]. To analyse the expression pat-terns in greater detail, northern blot hybridizations were carried out (Fig. 2). These experiments re-vealed that the expression of the five CCP genes under investigation was restricted to nodules. No hybridizing transcripts could be detected in unin-fected roots, leaves, seeds, epicotyls, stems and flowers (see Fig. 2), even after overexposure (data not shown). The mRNAs identified in nodules were about 450 bases long, as judged by RNA markers. Considering the very short poly(A) tails in the cDNAs isolated, these transcript lengths were in accordance to the CCP sequences pre-sented. As determined by cDNA-cDNA hybridiza-tions (data not shown) CCP transcripts were first

Fig. 1. Alignment of the deduced amino acid sequences of the broad bean late nodulins VfNOD-CCP1, VfNOD-CCP2, VfNOD-CCP3, VfNOD-CCP4 and VfNOD-CCP5, the late pea nodulin PsNOD6 [16] and the early pea nodulins PsENOD3 and PsENOD14 [35]. Amino acids identical in at least six of the eight sequences aligned are presented in reverse type. Shadowed amino acids indicate conservative substitutions. Putative signal peptide cleavage sites are marked by vertical arrows. Conserved cysteine residues are marked by arrowheads. Abbreviation: aa, amino acids.

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M.Fru¨hling et al./Plant Science152 (2000) 67 – 77

Fig. 2. Expression of CCP genes in different broad bean tissues. Northern blots containing 30mg of total RNA from root nodules, uninfected roots, leaves, seeds, epicotyls, stems and flowers, were hybridized against different CCP cDNA probes. The probes and the deduced length of hybridizing transcripts are indicated on the right. Abbreviation: kb, kilo-bases.

3.3. The CCP genes are expressed in central nodule tissues

To elucidate the spatial distribution of the CCP transcripts in broad bean root nodules, tissue print hybridizations were performed. Longitudinal sec-tions of mature nodules were printed onto nylon membranes and hybridized to CCP sense and anti-sense riboprobes. Representative results of these experiments are shown in Fig. 3. To relate hy-bridizing regions to distinct nodule zones we

visu-alized the interzone II/III in the nodule sections

used for printing by staining for starch (see Fig. 3B) [44]. In general, hybridization signals occured exclusively with the antisense probes (data not shown) and were found to be restricted to the central tissues of the nodule (Fig. 3A). No hy-bridization was detected in peripheral tissues or in the nodule meristem. CCP1, VfNOD-CCP3, VfNOD-CCP4 and VfNOD-CCP5

tran-scripts were located in the interzone II/III and the

nitrogen fixing zone III. VfNOD-CCP1 transcripts were additionally detectable in the ineffective zone IV, whereas detection of VfNOD-CCP4 tran-scripts extended to the distal region of the nodule and comprised large areas of the prefixing zone II. VfNOD-CCP2 transcripts were predominantly

present in the interzone II/III. In much lower

amounts hybridizing VfNOD-CCP2 transcripts were also found to be dispersed in the distal region of the nitrogen fixing zone III.

3.4. Genomic organsisation of VfNOD-CCP1 As a step towards the characterization of the genomic organization of CCP nodulin genes, we isolated a 20 kb VfNOD-CCP1 fragment from a genomic broad bean library. Subcloning resulted

in the identification of a 4.4 kb EcoRI/SalI

frag-ment in clone c2 – 19, which hybridized to a

VfNOD-CCP1 cDNA. Fig. 4 shows the sequence

of a 2424 bp EcoRI/SphI subfragment of this

clone. Sequence analysis revealed that the

VfNOD-CCP1 gene consisted of two exons that showed sequence identity to the VfNOD-CCP1 transcript sequence determined. The two exons were interrupted by an 99 bp intron separating the first exon encoding the secretory signal peptide from the second exon encoding the cysteine cluster

domain (see Fig. 4). The exon/intron boundaries

identified were in good agreement with the

consen-detectable inRhizobium infected broad bean roots

uniformly 7 days after inoculation. The expression of leghemoglobin genes starts earlier at day 6 after inoculation. Due to the nodule specificity and the onset of CCP expression during nodule formation, the polypeptides encoded by the CCP genes were classified as late nodulins, according to Nap and Bisseling [24,25].

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sus sequences of splice junctions in genes of di-cotyledonous plants [13]. Upstream of the first base corresponding to the full length VfNOD-CCP1 transcript sequence, the sequence most simi-lar to a consensus of sequences surrounding nodulin gene TATA boxes could be identified at

position −37 to −25 (ACACTATAAATTG, 10

out of 13 bases matched the consensus reported by Joshi [15]). In addition, an analysis of the

com-plete −1759/ +1 putative promoter region of the

VfNOD-CCP1 gene revealed the multiple presence

of the sequence motifs AAAGAT and CTCTT, characteristic of leghemoglobin and other nodulin gene promoters [34,39], on both strands of the DNA sequence (see Fig. 4). The CTCTT sequence

located from position −173 to −169 was

iden-tified as part of a short element, which resembled the OSE (organ-specific element) of the soybean leghemoglobin lbc3 gene promoter [37] both in sequence and position (Fig. 5A). Apart from these motifs similar to published promoter motifs, a 12 bp inverted repeat and a 17 bp direct repeat were

Fig. 3. Localization of transcripts coding for CCP nodulins in broad bean root nodules. (A) Representative tissue print hybridizations of longitudinal nodule sections against DIG-labelled antisense ribroprobes are shown for VfNOD-CCP1, VfNOD-CCP2, VfNOD-CCP3, VfNOD-CCP4 and VfNOD-CCP5. (B) Microphotographs of the nodule sections used for tissue printing after staining for starch. Cells of the interzone II/III stained black. In addition, starch was detected in the basal region of the nodules, which presumably corresponds to the senescence zone IV. (C) Diagrams of the distribution of hybridizing transcripts in the model nodule were concluded from a comparison of A and B. In the diagrams the nodule zones meristem, preinfection zone II, interzone II/III, nitrogen fixing zone III and ineffective zone IV are displayed from the top to the base.

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M.Fru¨hling et al./Plant Science152 (2000) 67 – 77

Fig. 4. Sequence of a 2424 bpEcoRI/SphI fragment from the genomic VfNOD-CCP1 clone c2 – 19. The deduced amino acid sequence of VfNOD-CCP1 is printed in bold type above the DNA sequence with the extent of a secretory signal peptide being underlined. The putative signal peptide cleavage site is marked by an vertical arrow. An intron interrupting the VfNOD-CCP1 coding region is displayed in small letters. The first base corresponding to the full length VfNOD-CCP1 transcript sequence is indicated by an dot and was chosen as position +1. In the upstream region, the putative TATA box sequence is underlined twice. The sequence element resembling leghemoglobin OSE sequences are shadowed, while nodulin consensus sequences of the type AAAGAT and CTCTT on both strands of the DNA are underlined. A inverted repeat and a direct repeat are marked by arrows below the sequence.

located on the VfNOD-CCP1 putative promoter (Fig. 5B). The significance of these motifs for theactivity of the putative VfNOD-CCP1 pro-moter has to be demonstrated by the analysis of

promoter-reporter-gene fusions in transgenic

plants.

4. Discussion

In this study, we reported on the isolation and characterization of five transcript sequences from broad bean root nodules encoding different mem-bers of a polypeptide family. Northern blot

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exper-iments indicated that these transcripts occur exclu-sively in nodules. Hybridizing mRNAs were not detected in any other broad bean tissue tested. Taking also into account the first appearance of the transcripts in nodules 1 day after the onset of leghemoglobin expression the corresponding gene products can be regarded as late nodulins.

The amino acid sequences deduced from the broad bean transcript revealed homologies to a family of pea nodulins including the early nodulins PsENOD3 and PsENOD14 [35] and the late nodulin PsNOD6 [16]. Whereas in general the overall identities between the sequences are quite low, a bipartite domain structure of the broad bean and the pea nodulins was found to be well conserved, indicating a common ancestry of these nodulins. All of them are small polypeptides of about 7 kDa containing a N-terminal hydrophobic region probably serving as a secretory signal pep-tide for targeting to the plant endomembrane sys-tem. The common feature of the processed polypeptides representing the second domain are four cysteine residues which are arranged in two clusters. Considering these cysteine clusters we designated the five late nodulins VfNOD-CCP1,

VfNOD-CCP2, VfNOD-CCP3, VfNOD-CCP4

and VfNOD-CCP5 according to the nomenclature rules for nodulins [24,25,42]. It is obvious to spec-ulate that the different members of the CCP nodulin families from both pea and broad bean have similar functions in root nodules close related to their highly conserved cysteine residues. Since the spatial distribution of these residues was also

found in metal binding proteins [1], Scheres et al. [35] assumed a role for PsENOD3 and PsENOD14 in metal transport. A similar function was pro-posed for members of a soybean nodulin family characterized by the prescence of a putative signal peptide and the existence of two conserved

do-mains containing each two Cys-X7-Cys motifs

[32,34]. Apart from the correlation in structural features no sequence homologies were found

be-tween the soybean nodulins and the CCP

nodulins. However, whether or not the broad bean, pea and soybean nodulins mentioned are able to bind metal ions remains to be established biochemically. In any case, the supply of metal ions to the microsymbiont by the plant must be

part of the Rhizobium-legume interaction. The

bacteroids which for example require cobalt for the synthesis of vitamin B12 [7] and high amounts of molybdenum and iron for the synthesis of nitrogenase [22] totally depend on the plant for nutrition. Therefore, the existence of nodule-spe-cific metal transport proteins would be expected. They could be part of the peribacteroid space which constitute the symbiotic interface or could contribute to metal storage in the vacuoles of infected nodule cells. Interestingly, the early nodulin genes PsENOD3 and PsENOD14 and the late nodulin gene PsNOD6 are only expressed in infected cells [16,35]. In mature nodules the PsNOD6 mRNA is first detectable at the begin-ning of the interzone II – III and this transcript is present at a constant level in the older cell layers of the central tissue. A comparable spatial

expres-Fig. 5. Sequences of putative VfNOD-CCP1 promotor elements. (A) Sequence of the soybean Gmlbc3 organ-specific element [39] as compared to thesesbania rostrataSrlb3 nodule-infected-cell-expression (NICE) element [40] and a similar element found in the putative VfNOD-CCP1 promotor. The nodulin motifs AAAGAT and CTCTT are boxed. (B) Sequence of a 12 bp inverted repeat and a 17 bp direct repeat located on the putative VfNOD-CCP1 promotor.

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M.Fru¨hling et al./Plant Science152 (2000) 67 – 77

sion pattern was observed for the broad bean nodulin genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 whereas the VfNOD-CCP4 gene and the early nodulins genes PsENOD3 and PsENOD14 are first induced in the prefixing zone II [35]. The distribution of the CCP transcripts VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 hints towards the requirement of the encoded nodulins in later stages of nodule development, whereas the VfNOD-CCP4 gene product might additionally be related to the infection process. In contrast to all other CCP nodulins tested a strong expression of the VfNOD-CCP2 gene only could be detected the interzone II – III region. Therefore the VfNOD-CCP2 gene could be regarded as a marker gene for the interzone II – III region in broad bean nodules like the previously described early nodulin gene VfENOD-GRP3 [20].

Considering the differences found in the spatial expression patterns of the CCP genes we assume that their regulation involves different mecha-nisms. The isolation of the VfNOD-CCP1 gene and its putative promoter region was a first step towards an analysis of the regulation of CCP gene expression. Sequence analysis of the genomic VfNOD-CCP1 fragment revealed the presence of an intron seperating two exons encoding the puta-tive signal peptide and the cysteine cluster domain of the VfNOD-CCP1 polypeptide. The same ge-nomic organisation resembling the bipartite do-main structure of the CCP nodulins was also found in the PsNOD6 gene [16], which further underlines the common ancestry of these nodulins. A number of sequence elements involved in medi-ating nodule specific expression have already been identified for several late nodulin gene promoters [3,4]. Computer searches for these motifs within the VfNOD-CCP1 promoter region identified mul-tiple copies of the common nodulin sequences AAAGAT and CTCTT. As is evident from Fig. 5A, the typical arrangement of AAAGAT and CTCTT subelements found in the soybean lbc3 OSE [39] and the Sesbania glb3 NICE elements [40] was fulfilled by one sequence motif, although the AAAGAT sequence itself was not perfectly conserved. Ramlov et al. [31] and Szczyglowski et al. [40] demonstrated that the CTCTT motif were imperative for the function of these elements, whereas mutations in the AAAGAT sequence had less pronounced effects. This observation could explain the lack of conservation of the AAAGAT

subsequence in the corresponding VfNOD-CCP1 promoter element. However, to assess the rele-vance of all sequence elements identified on the DNA level for the nodule specific expression of the VfNOD-CCP1 gene, further experiments using promoter-reporter-gene fusions in transgenic nod-ules have to be performed. To investigate the properties and functions of the broad bean CCP nodulins identified in more detail, we intend to characterize these polypeptides biochemically.

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[11] A.M. Frischauf, H. Lehrach, A. Poustka, N. Murray, Lambda replacement vectors carrying polylinker se-quences, J. Mol. Biol. 170 (1983) 827 – 842.

[12] A. Fru¨hling, H. Roussel, V. Gianinazzi-Pearson, A. Pu¨h-ler, A.M. Perlick, The Vicia faba leghemoglobin gene

VfLb29 is induced in root nodules and in roots colonized by the arbuscular mycorrhizal fungusGlomus fascicula

-tum, Mol. Plant Microbe Interact. 10 (1997) 124 – 131. [13] B.A. Hanley, M.A. Schuler, Plant intron sequences:

evi-dence for distinct groups of introns, Nucleic Acids Res. 16 (1988) 7159 – 7176.

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[14] D.G. Higgins, P.M. Sharp, CLUSTAL: a package for performing multiple sequence alignment on a microcom-puter, Gene 73 (1988) 237 – 244.

[15] C.P. Joshi, An inspection of the domain between putative TATA box and translation start site in 79 plant genes, Nucleic Acids Res. 15 (1987) 6643 – 6653.

[16] I. Kardailsky, W.-C. Yang, A. Zalensky, A. van Kammen, T. Bisseling, The pea late noduling gene PsNOD6 is homologous to the early nodulin genes PsENOD3/14 and is expressed after the leghaemoglobin genes, Plant Mol. Biol. 23 (1993) 1029 – 1037.

[17] C. Kessler, Nonradioactive Labeling and Detection of Biomolecules, Springer-Verlag, Berlin, 1992.

[18] J.W. Kijne, The Rhizobium infection process, in: G. Stacey, R.H. Burris, J. Hardd (Eds.), Biological Nitrogen Fixation, Chapman and Hall, New York, 1992, pp. 349 – 398.

[19] H. Ku¨ster, A.M. Perlick, A. Pu¨hler, Members of a broadbean nodulin family with partial homologies to the alfalfa nodulin 25 are composed of two types of amino acid repeats flanked by unique amino acid sequence termini, Plant Mol. Biol. 24 (1994) 143 – 157.

[20] H. Ku¨ster, G. Schro¨der, M. Fru¨hling, U. Pich, M. Rieping, I. Schubert, A.M. Perlick, A. Pu¨hler, The nod-ule-specificVfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I ofVicia fabaL. and is predominantly expressed in the interzone II – III of root nodules, Plant Mol. Biol. 28 (1995) 405 – 421. [21] H. Ku¨ster, M. Fru¨hling, A. Pu¨hler, A.M. Perlick, The

modular nodulins Nvf-28/32 of broad bean (Vicia faba

L.): alternative exon combinations account for different modular structures, Mol. Gen. Genet. 252 (1996) 648 – 657.

[22] T. Ljones, The enzyme system, in: A. Quispel (Ed.), Biology of Nitrogen Fixation, North-Holland Publishing Company, Amsterdam, 1974, pp. 617 – 639.

[23] P. Mylona, K. Pawloski, T. Bisseling, Symbiotic nitrogen fixation, Plant Cell 7 (1995) 869 – 885.

[24] J.P. Nap, T. Bisseling, Developmental biology of a plant-prokaryote symbiosis: the legume root nodule, Science 250 (1990) 948 – 954.

[25] J.P. Nap, T. Bisseling, The roots of nodulins, Physiol. Plant. 79 (1990) 404 – 414.

[26] H. Nielsen, J. Engelbrecht, S. Brunak, G. von Heijne, Identification of prokaryotic and eukaryotic signal pep-tides and prediction of their cleavage sites, Protein Eng. 10 (1997) 1 – 6.

[27] W.R. Pearson, D.J. Lipman, Improved tools for biologi-cal sequence comparison, Proc. Natl. Acad. Sci. USA 85 (1988) 2444 – 2448.

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[29] A.M. Perlick, M. Fru¨hling, G. Schro¨der, S.C. Frosch, A. Pu¨hler, The broad bean gene VfNOD32 encodes a nodulin with sequence similarities to chitinases that is homologous to (a/b)8-barrel-type seed proteins, Plant Physiol. 110

(1996) 147 – 154.

[30] U.B. Priefer, Genes involved in lipopolysaccharide pro-duction and symbiosis are clustered on the chromosome

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3.3. The CCP genes are expressed in central

nodule tissues

To elucidate the spatial distribution of the CCP

transcripts in broad bean root nodules, tissue print

hybridizations were performed. Longitudinal

sec-tions of mature nodules were printed onto nylon

membranes and hybridized to CCP sense and

anti-sense riboprobes. Representative results of these

experiments are shown in Fig. 3. To relate

hy-bridizing regions to distinct nodule zones we

visu-alized the interzone II

/

III in the nodule sections

used for printing by staining for starch (see Fig.

3B) [44]. In general, hybridization signals occured

exclusively with the antisense probes (data not

shown) and were found to be restricted to the

central tissues of the nodule (Fig. 3A). No

hy-bridization was detected in peripheral tissues or in

the nodule meristem. CCP1,

VfNOD-CCP3, VfNOD-CCP4 and VfNOD-CCP5

tran-scripts were located in the interzone II

/

III and the

nitrogen fixing zone III. VfNOD-CCP1 transcripts

were additionally detectable in the ineffective zone

IV, whereas detection of VfNOD-CCP4

tran-scripts extended to the distal region of the nodule

and comprised large areas of the prefixing zone II.

VfNOD-CCP2 transcripts were predominantly

present in the interzone II

/

III. In much lower

amounts hybridizing VfNOD-CCP2 transcripts

were also found to be dispersed in the distal region

of the nitrogen fixing zone III.

3.4. Genomic organsisation of VfNOD

-

CCP

1 As a step towards the characterization of the

genomic organization of CCP nodulin genes, we

isolated a 20 kb VfNOD-CCP1 fragment from a

genomic broad bean library. Subcloning resulted

in the identification of a 4.4 kb

Eco

RI

/Sal

I

frag-ment in clone

c

2 – 19, which hybridized to a

VfNOD-CCP1 cDNA. Fig. 4 shows the sequence

of a 2424 bp

Eco

RI

/Sph

I subfragment of this

clone.

Sequence

analysis

revealed

that

the

VfNOD-CCP1 gene consisted of two exons that

showed sequence identity to the VfNOD-CCP1

transcript sequence determined. The two exons

were interrupted by an 99 bp intron separating the

first exon encoding the secretory signal peptide

from the second exon encoding the cysteine cluster

domain (see Fig. 4). The exon

/

intron boundaries

identified were in good agreement with the

consen-detectable in

Rhizobium

infected broad bean roots

uniformly 7 days after inoculation. The expression

of leghemoglobin genes starts earlier at day 6 after

inoculation. Due to the nodule specificity and the

onset of CCP expression during nodule formation,

the polypeptides encoded by the CCP genes were

classified as late nodulins, according to Nap and

Bisseling [24,25].

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cotyledonous plants [13]. Upstream of the first

base corresponding to the full length

VfNOD-CCP1 transcript sequence, the sequence most

simi-lar to a consensus of sequences surrounding

nodulin gene TATA boxes could be identified at

position

−

37 to

−

25 (ACACTATAAATTG, 10

out of 13 bases matched the consensus reported by

Joshi [15]). In addition, an analysis of the

com-plete

−

1759

/ +

1 putative promoter region of the

VfNOD-CCP1 gene revealed the multiple presence

characteristic of leghemoglobin and other nodulin

gene promoters [34,39], on both strands of the

DNA sequence (see Fig. 4). The CTCTT sequence

located from position

−

173 to

−

169 was

iden-tified as part of a short element, which resembled

the OSE (organ-specific element) of the soybean

leghemoglobin lbc3 gene promoter [37] both in

sequence and position (Fig. 5A). Apart from these

motifs similar to published promoter motifs, a 12

bp inverted repeat and a 17 bp direct repeat were

(3)

located on the VfNOD-CCP1 putative promoter

(Fig. 5B). The significance of these motifs for

theactivity of the putative VfNOD-CCP1

pro-moter has to be demonstrated by the analysis of

promoter-reporter-gene

fusions

in

transgenic

plants.

4. Discussion

In this study, we reported on the isolation and

characterization of five transcript sequences from

broad bean root nodules encoding different

mem-bers of a polypeptide family. Northern blot

(4)

exper-sively in nodules. Hybridizing mRNAs were not

detected in any other broad bean tissue tested.

Taking also into account the first appearance of

the transcripts in nodules 1 day after the onset of

leghemoglobin expression the corresponding gene

products can be regarded as late nodulins.

The amino acid sequences deduced from the

broad bean transcript revealed homologies to a

family of pea nodulins including the early nodulins

PsENOD3 and PsENOD14 [35] and the late

nodulin PsNOD6 [16]. Whereas in general the

overall identities between the sequences are quite

low, a bipartite domain structure of the broad

bean and the pea nodulins was found to be well

conserved, indicating a common ancestry of these

nodulins. All of them are small polypeptides of

about 7 kDa containing a N-terminal hydrophobic

region probably serving as a secretory signal

pep-tide for targeting to the plant endomembrane

sys-tem. The common feature of the processed

polypeptides representing the second domain are

four cysteine residues which are arranged in two

clusters. Considering these cysteine clusters we

designated the five late nodulins VfNOD-CCP1,

VfNOD-CCP2,

VfNOD-CCP3,

VfNOD-CCP4

and VfNOD-CCP5 according to the nomenclature

rules for nodulins [24,25,42]. It is obvious to

spec-ulate that the different members of the CCP

nodulin families from both pea and broad bean

have similar functions in root nodules close related

to their highly conserved cysteine residues. Since

the spatial distribution of these residues was also

[35] assumed a role for PsENOD3 and PsENOD14

in metal transport. A similar function was

pro-posed for members of a soybean nodulin family

characterized by the prescence of a putative signal

peptide and the existence of two conserved

do-mains containing each two Cys-X

-Cys motifs

[32,34]. Apart from the correlation in structural

features no sequence homologies were found

be-tween

the

soybean

nodulins

and

the

CCP

nodulins. However, whether or not the broad

bean, pea and soybean nodulins mentioned are

able to bind metal ions remains to be established

biochemically. In any case, the supply of metal

ions to the microsymbiont by the plant must be

part of the

Rhizobium

-legume interaction. The

bacteroids which for example require cobalt for

the synthesis of vitamin B12 [7] and high amounts

of molybdenum and iron for the synthesis of

nitrogenase [22] totally depend on the plant for

nutrition. Therefore, the existence of

nodule-spe-cific metal transport proteins would be expected.

They could be part of the peribacteroid space

which constitute the symbiotic interface or could

contribute to metal storage in the vacuoles of

infected nodule cells. Interestingly, the early

nodulin genes PsENOD3 and PsENOD14 and the

late nodulin gene PsNOD6 are only expressed in

infected cells [16,35]. In mature nodules the

PsNOD6 mRNA is first detectable at the

begin-ning of the interzone II – III and this transcript is

present at a constant level in the older cell layers

of the central tissue. A comparable spatial

(5)

sion pattern was observed for the broad bean

nodulin genes VfNOD-CCP1, VfNOD-CCP3 and

VfNOD-CCP5 whereas the VfNOD-CCP4 gene

and the early nodulins genes PsENOD3 and

PsENOD14 are first induced in the prefixing zone

II [35]. The distribution of the CCP transcripts

VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5

hints towards the requirement of the encoded

nodulins in later stages of nodule development,

whereas the VfNOD-CCP4 gene product might

additionally be related to the infection process. In

contrast to all other CCP nodulins tested a strong

expression of the VfNOD-CCP2 gene only could

be detected the interzone II – III region. Therefore

the VfNOD-CCP2 gene could be regarded as a

marker gene for the interzone II – III region in

broad bean nodules like the previously described

early nodulin gene VfENOD-GRP3 [20].

Considering the differences found in the spatial

expression patterns of the CCP genes we assume

that their regulation involves different

mecha-nisms. The isolation of the VfNOD-CCP1 gene

and its putative promoter region was a first step

towards an analysis of the regulation of CCP gene

expression. Sequence analysis of the genomic

VfNOD-CCP1 fragment revealed the presence of

an intron seperating two exons encoding the

puta-tive signal peptide and the cysteine cluster domain

of the VfNOD-CCP1 polypeptide. The same

ge-nomic organisation resembling the bipartite

do-main structure of the CCP nodulins was also

found in the PsNOD6 gene [16], which further

underlines the common ancestry of these nodulins.

A number of sequence elements involved in

medi-ating nodule specific expression have already been

identified for several late nodulin gene promoters

[3,4]. Computer searches for these motifs within

the VfNOD-CCP1 promoter region identified

mul-tiple copies of the common nodulin sequences

AAAGAT and CTCTT. As is evident from Fig.

5A, the typical arrangement of AAAGAT and

CTCTT subelements found in the soybean lbc3

OSE [39] and the Sesbania glb3 NICE elements

[40] was fulfilled by one sequence motif, although

the AAAGAT sequence itself was not perfectly

conserved. Ramlov et al. [31] and Szczyglowski et

al. [40] demonstrated that the CTCTT motif were

imperative for the function of these elements,

whereas mutations in the AAAGAT sequence had

less pronounced effects. This observation could

explain the lack of conservation of the AAAGAT

subsequence in the corresponding VfNOD-CCP1

promoter element. However, to assess the

rele-vance of all sequence elements identified on the

DNA level for the nodule specific expression of

the VfNOD-CCP1 gene, further experiments using

promoter-reporter-gene fusions in transgenic

nod-ules have to be performed. To investigate the

properties and functions of the broad bean CCP

nodulins identified in more detail, we intend to

characterize these polypeptides biochemically.

References

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evi-dence for distinct groups of introns, Nucleic Acids Res. 16 (1988) 7159 – 7176.

(6)

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